The prototypic Arenavirus lymphocytic choriomeningitis virus (LCMV) is an excellent model to study the molecular and cellular biology of hemorrhagic fever arenaviruses, including Lassa fever virus (LFV). These viruses cause severe human disease, and they pose a real threat as agents of bioterrorism. We have developed a reverse genetic system for LCMV. We can now probe the function of viral genes in cell entry, virus replication, assembly and budding. We have obtained evidence that Arenavirus Z protein is the main driving force of virus budding. Arenavirus Z proteins contain proline-rich motifs PPXY and PT/SAP, which have been identified as essential for budding of several viruses including HIV-1 and Ebola. Evidence indicates that many enveloped viruses are capable of hijacking the cellular multivesicular body (MVB) machinery to escape the cell. We hypothesize that budding of LFV is mediated by the interaction of Z proline-rich domains with specific host factors, including components of the MVB pathway. Understanding these interactions will facilitate targeting of these virally reprogrammed host cellular processes as a new strategy to combat arenaviruses. We propose two specific aims. First, to determine the role of PPPY and PTAPP motifs in LFV Z mediated budding. Z proteins with mutations in PPPY or PTAPP, or both, motifs will be evaluated for their competence to promote budding of virus like particles (VLPs). Second, to identify host factors required for Arenavirus budding. We will test the hypothesis that TSG101 and Nedd4 proteins, both members of the MVB pathway, interact with LFV Z and that these interactions are relevant in LFV Z mediated budding. We will use the tandem affinity purification (TAP) method combined with mass spectrometry procedures to identify other Z-interacting cellular proteins. We will use biochemical and cellular assays to characterize Z/host factor interactions. The functional significance of these host factors in Arenavirus budding will be investigated by quantifying budding of VLPs in cells where expression of the host protein of interest has been disrupted by RNA interference.